Method of decreasing liquiritigenin-derived isoflavones relative to total isoflavones in plants and plants producing reduced ratio of liquiritigenin-derived isoflavones relative to total isoflavones

ABSTRACT

This invention pertains to methods for decreasing the ratio of liquiritigenin-derived isoflavones relative to total isoflavone levels in isoflavonoid-producing plants and plant parts by transforming plants with a recombinant DNA construct comprising a nucleic acid sequence of at least 200 nucleotides and having at least 75% sequence identity to a polynucleotide encoding a chalcone reductase. More preferably, this invention pertains to methods for decreasing the ratios of daidzein and glycitein and their conjugates relative to total isoflavone levels in soybean plants and soybean plant parts by transforming plants with a recombinant DNA construct comprising a nucleic acid sequence of at least 200 nucleotides and having at least 75% sequence identity to a polynucleotide encoding a chalcone reductase.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/433,433, filed Dec. 13, 2003. The entire content ofthis application is hereby incorporated by reference.

[0002] This invention relates to altering isoflavone levels in anisoflavonoid-producing plant and, in particular, to a method fordecreasing the ratio of liquiritigenin-derived isoflavones relative tototal isoflavone levels in isoflavonoid-producing plants and plant partsby transforming plants with a recombinant DNA construct comprising anucleic acid sequence of at least 200 nucleotides and having at least75% sequence identity to a polynucleotide encoding a chalcone reductase.

[0003] Isoflavonoids represent a class of secondary metabolites producedpredominantly in legumes by a branch of the phenylpropanoid pathway andinclude such compounds as isoflavones, isoflavanones, rotenoids,pterocarpans, isoflavans, quinone derivatives,3-aryl-4-hydroxycoumarins, 3-arylcoumarins, isoflav-3-enes, coumestans,alpha-methyldeoxybenzoins, 2-arylbenzofurans, isoflavanol,coumaronochromone and the like. In plants, these compounds are known tobe involved in interactions with other organisms and to participate inthe defense responses of legumes against phytopathogenic microorganisms(Dewick, P. M. (1993) in The Flavonoids, Advances in Research Since1986, Harborne, J. B. Ed., pp. 117-238, Chapman and Hall, London).Isoflavonoid-derived compounds also are involved in symbioticrelationships between roots and rhizobial bacteria that eventuallyresult in nodulation and nitrogen-fixation (Phillips, D. A. (1992) inRecent Advances in Phytochemistry. Vol. 26, pp 201-231, Stafford, H. A.and Ibrahim, R. K., Eds, Plenum Press, New York), and overall they havebeen shown to act as antibiotics, repellents, attractants, and signalcompounds (Barz, W. and Welle, R. (1992) Phenolic Metabolism in Plants,pp 139-164, Ed by H. A. Stafford and R. K. Ibrahim, Plenum Press, NewYork).

[0004] Isoflavonoids have also been reported to have physiologicalactivity in animal and human studies. For example, it has been reportedthat the isoflavones found in soybean seeds possess antihemolytic (Naim,M., et al. (1976) J. Agric. Food Chem. 24:1174-1177), antifungal (Naim,M., et al. (1974) J. Agr. Food Chem. 22:806-810), estrogenic (Price, K.R. and Fenwick, G. R. (1985) Food Addit. Contam. 2:73-106),tumor-suppressing (Messina, M. and Bames, S. (1991) J. Natl. CancerInst. 83:541-546; Peterson, G., et al. (1991) Biochem. Biophys. Res.Commun. 179:661-667), hypolipidemic (Mathur, K., et. al. (1964) J. Nutr.84:201-204), and serum cholesterol-lowering (Sharma, R. D. (1979) Lipids14:535-540) effects. In addition, both epidemiological anddietary-intervention studies indicate that when isoflavones in soybeanseeds and in subsequent protein products prepared from the seeds arepart of the human dietary intake, those products provide manysignificant health benefits (Messina, M. J. (1999) Am. J. Clin. Nutr.70:439S-450S; Walker, C. L. (2002) Recent Prog. Horm. Res.57:277-294;Davis, S. R. et al. (1999) Recent Prog. Horm. Res. 54, 185-210; Messina,M. J. (1999) Am. J. Clin. Nutr. 70 (suppl): 439S-450S; Watanabe, S. etal. (2002) Biomed. Pharmacother. 56, 302-312; Clarkson, T. B. (2002) J.Nutr. 132:566S-569S).

[0005] Soybean seeds contain three types of isoflavone aglycones:daidzein, genistein, and glycitein. However, free isoflavones rarelyaccumulate to high levels in soybeans; instead they are usuallyconjugated to carbohydrates or organic acids. Each aglycone can be foundin three different forms: glucoside conjugates known as daidzin,genistin, and glycitin; malonylglucoside conjugates known as6″-O-malonyldaidzin, 6″-O-malonylgenistin and 6″-O-malonylglycitin.During processing acetylglucoside conjugates known as6′-O-acetyidaidzin, 6′-O-acetylgenistin, and 6′-O-acetylglycitin aresometimes produced.

[0006] The total isoflavone levels as well as the distribution amongdifferent aglycones is quite variable in soybean seeds and is affectedby both genetics and environmental conditions such as growing locationand temperature during seed fill (Tsukamoto, C., et al. (1995) J. Agric.Food Chem. 43:1184-1192; Wang, H. and Murphy, P. A. (1994) J. Agric.Food Chem. 42:1674-1677). In addition, isoflavonoid content in legumescan be stress-induced by pathogen attack, wounding, high UV lightexposure and pollution (Dixon, R. A. and Paiva, N. L. (1995) Plant Cell7:1085-1097). The genistein isoflavonoid forms make up the most abundantgroup in most food products, while the daidzein and the glycitein formsare present in lower levels (Murphy, P. A. (1999) J. Agric. Food Chem.47:2697-2704).

[0007] The biosynthetic pathway for isoflavonoids in soybean and theirrelationship with several other classes of phenylpropanoids is presentedin FIG. 1. Production of coumaryl-CoA from phenylalanine requiresphenylalanine ammonia lyase to convert phenylalanine to cinnamate,cinnamic acid hydroxylase to convert cinnamate to p-coumarate, andcoumarate:CoA ligase to convert p-coumarate to p-coumaroyl-CoA. Ligninsmay be produced from p-coumaroyl-CoA or from p-coumarate. Soybeanchalcone synthase catalyzes the conversion of p-coumaroyl-CoA to 4, 2′,4′, 6′-tetrahydroxychalcone that is isomerized in a reaction catalyzedby chalcone isomerase to naringenin, the precursor to genistein,flavones, flavonols, and others. Alternatively, chalcone reductasetogether with a chalcone synthase and NADPH as a cofactor, act in theformation of isoliquiritigenin which is then isomerized to formliquiritigenin, the precursor to daidzein, glycitein, and thepterocarpan phytoalexins (Welle, R. and Grisebach, H. (1988) FEBS Lett.236:221-225). Chalcone reductase is also known as deoxychalcone synthaseand is abbreviated CHR. Polynucleotide sequences encoding CHR have beenreported for soybean (Glycine max; NCBI General Identifier No. 99953),kudzu vine (Pueraria montana var. lobata; NCBI General Identifier No.20147510), and alfalfa (Medicago sativa subsp. sativa; NCBI GeneralIdentifier No. 537298), as well as others. Glycitein synthesis is notyet clearly defined, but is likely made from liquiritigenin(Latunde-Dada, A. O. et al. (2001) J. Biol. Chem. 276,1688-1685).Genistein synthesis shares the naringenin intermediate with theflavonol/anthocyanin branch of the phenylpropanoid pathway. In all casesthe unique aryl migration reaction to create the isoflavones is mediatedby isoflavone synthase, or IFS. Sequences encoding the IFS gene havebeen identified for licorice (Akashi, T. et al. (1999) Plant Physiol.121:821-828) and soybean (Steele, C. L. (1999) Arch Biochem. Biophys.367:146-150; Jung, W. et al. (2000) Nature Biotech. 18:208-212; editor'scorrection: Nature Biotech. 18:559).

[0008] The use of sequences with low homology to CHR to controlstrawberry ripening is suggested in PCT publication No. WO 97/27295(published Jul. 31, 1997). In the publication 27 ripening-related cloneswere selected and compared with sequences in the EMBL database using theGCG software. One of these clones was identified as having homology to apolynucleotide sequence encoding CHR. No additional information is givenabout the strawberry CHR or its use in regulating fruit ripening.However strawberry does not make isoflavonoids and would therefore notbe expected to make CHR.

[0009] The physiological benefits associated with isoflavonoids in bothplants and humans make the manipulation of their contents in crop plantshighly desirable. The human body responds differently to differentisoflavones. Although both daidzein and genistein act as phytoestrogens,genistein seems to have other specific abilities such as DNAtopoismerase and tyrosine protein kinase as well as antioxidant and cellcycle inhibitor activity (Dixon, R. A. and Ferreira, D. (2002)Phytochemistry 60:205-211). At times it may be desirable to consumegenistein and not daidzein.

[0010] The flavor of soybeans is affected by multiple factors. Amongthese are isoflavones, and in particular daidzin and genistin. Okubo etal. identified the compounds in soybean that produce a dry mouth feeland reported a quantitative measurement (Okubo et al. (1992) Biosci.Biotech. Biochem. 56:99-103). In this study the concentration at whichthe dry mouth feel was first detected was reported as the thresholdvalue. The threshold value for detection of daidzin was found to be 10⁻⁵to 10⁻⁶ M while the threshold value for daidzein, its aglycone, was 10⁻⁶M. The threshold value for genistin was 10⁻⁵ M and the one for genisteinwas 10⁻⁴ M. Thus, reduction of the levels of daidzin and its aglyconewithout effecting the levels of genistin or its aglycone will result insoybeans with better flavor and still improved health benefits. BecauseCHR catalyzes the committed step in the production ofliquiritigenin-derived isoflavones, suppression of this enzyme willresult in soybeans with lower levels of liquiritigenin-derivedisoflavones without affecting the naringenin-derived isoflavones.

SUMMARY OF THE INVENTION

[0011] The present invention includes a method for decreasing the ratioof liquiritigenin-derived isoflavones relative to total isoflavonelevels in an isoflavonoid-producing plant the method comprising: a)transforming a plant cell with a recombinant construct comprising apromoter operably linked to a nucleic acid sequence of at least 200nucleotides having at least 75% sequence identity to SEQ ID NO:4; b)regenerating a transformed plant from the transformed plant cell of (a);and c) evaluating the transformed plant obtained from step (b) for areduced ratio of liquiritigenin-derived isoflavones relative to totalisoflavone levels as compared to the ratio of liquiritigenin-derivedisoflavones relative to total isoflavone levels in an untransformedplant.

[0012] In a second embodiment, this invention includes anisoflavonoid-producing plant made by the method of the invention whereinthe plant has a reduced ratio of liquiritigenin-derived isoflavonesrelative to total isoflavone levels as compared to the ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsin an untransformed plant. Isoflavonoid-producing plants of interestinclude soybean, clover, mung bean, lentil, hairy vetch, alfalfa,lupine, sugar beet, and snow pea. Seeds or plant parts of such plantsare also of interest.

[0013] In a third embodiment, this invention includesisoflavonoid-containing products such as protein isolate, proteinconcentrate, meal, grits, full fat and defatted flours, texturedproteins, textured flours, textured concentrates, textured isolates,soymilk, tofu, fermented soy products, and whole bean soy products whichare obtained from seeds or plant parts of the invention.

[0014] In a fourth embodiment, this invention includes a food,nutritional supplement, food bar, or beverage which has incorporatedtherein an isoflavonoid-containing product of the invention.

[0015] In a fifth embodiment, this invention includes anisoflavonoid-producing plant comprising in its genome a recombinantconstruct comprising a promoter operably linked to a nucleic acidsequence of at least 200 nucleotides and having at least 75% sequenceidentity to SEQ ID NO:4 wherein the plant has a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsas compared to the ratio of liquiritigenin-derived isoflavones relativeto total isoflavone levels in an untransformed plant.

[0016] In a sixth embodiment, this invention includes a method ofproducing an isoflavonoid-containing product having a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelswhich comprises: (a) cracking the seeds of the invention to remove themeats from the hulls; and (b) flaking the meats obtained in step (a) toobtain the desired flake thickness.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0017] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0018]FIG. 1 depicts the soybean biosynthetic pathway from phenylalanineto glycitein, daidzein, genistein, and dihydroflavonol.

[0019]FIG. 2 depicts a representation of the seed-specific expressionvector pKS151. The two copies of the 36-nucleotide sequence areindicated by “EL”, and the inverted-repeat of the 36-nucleotidesequences is indicated by “(EL)”.

[0020]FIGS. 3 and 4 depict the percentage of total isoflavones that thesum of daidzin and glycitin represent in bulk R1 seeds from transformedplants containing the CHR construct. This percentage was determinedusing the equation: 100((daidzin+glycitin)/total isoflavones).

[0021] The following sequence descriptions and Sequences Listingattached hereto comply with the rules governing nucleotide and/or aminoacid sequence disclosures in patent applications as set forth in 37C.F.R. §1.821-1.825. The Sequence Listing contains the one letter codefor nucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

[0022] SEQ ID NO:1 is the nucleotide sequence of plasmid pKS151 aseed-specific gene silencing vector.

[0023] SEQ ID NO:2 is the nucleotide sequence of primer chalconereductase-Not1-sense, used to amplify a portion of the gene encodingchalcone reductase in clone src3c.pk009.e4.

[0024] SEQ ID NO:3 is the nucleotide sequence of primer chalconereductase-Not1-antisense, used to amplify a portion of the gene encodingchalcone reductase in clone src3c.pk009.e4.

[0025] SEQ ID NO:4 is the nucleotide sequence of a contig comprising thecDNA insert in plasmid src3c.pk009.e4 as well as other soybean ESTs.Nucleotides 270 through 1214 are the coding region for a chalconereductase.

[0026] SEQ ID NO:5 is the nucleotide sequence of primer 3, used todetect the presence of the chalcone reductase construct in transformedplants.

[0027] SEQ ID NO:6 is the nucleotide sequence of primer 4, used todetect the presence of the chalcone reductase construct in transformedplants.

[0028] SEQ ID NO:7 is the nucleotide sequence of nucleotides 5451-5567from SEQ ID NO:1. This nucleotide sequence corresponds to thepolynucleotide fragment consisting essentially of a unique Not 1restriction endonuclease site surrounded by nucleotides that promoteformation of a stem structure which are flanked by Eag I restrictionendonuclease sites.

[0029] SEQ ID NO:8 is the amino acid sequence that would result fromtranslating one 30-nucleotide sequence repeat in plasmid pKS151, forexample, from nucleotides 5457-5486 of SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION

[0030] All patents, patent applications, and publications cited areincorporated herein by reference in their entirety.

[0031] In the context of this disclosure, a number of terms shall beutilized.

[0032] The term “isoflavonoid(s)”refers to a large group of polyphenoliccompounds, based on a common diphenylpropane skeleton, which occurnaturally in plants. “Isoflavones” are the most abundant of the naturalisoflavonoids with over 160 isoflavone aglycones now recognized. Thisterm, as used herein, includes, but is not limited to, the three typesof isoflavone aglycones found in soybean daidzein, genistein, andglycitein. Free isoflavones rarely accumulate to high levels insoybeans; instead they are usually conjugated to carbohydrates ororganic acids. The term isoflavones includes all of the conjugates suchas the glucoside conjugates daidzin, genistin, and glycitin;malonylglucoside conjugates known as 6″-O-malonyldaidzin,6″-O-malonylgenistin, and 6″-O-malonylglycitin; and acetylglucosideconjugates known as 6′-O-acetyldaidzin, 6′-O-acetylgenistin, and6′-O-acetylglycitin that are sometimes produced during processing.

[0033] “Liquiritigenin-derived isoflavones” as used herein refers tothose isoflavones produced in a branch of the phenylpropanoid pathwayinvolving a chalcone reductase-mediated reaction. Examples ofliquiritigenin-derived isoflavones include, but are not limited to,daidzein, formononetin, isoformononetin, dimethyidaidzein,7,2′,4′-trihydroxyisoflavone, 2′-hydroxyformononetin, theralin,3′hydroxydaidzein, 3′-hydroxyformononetin (calycosin), sayanedin,cabreuvin, pseudobaptigenin, 7-methoxy-3′4′-methylenedioxyisoflavone,koparin, 2-hydroxy-7,3′,4′-trimethoxyisoflavone, glyzaglabrin,7,2′,4′,5′-tetramethoxyisoflavone, gliricidin, texasin, glyitein,kakkatin, aformosin, odoratin, cladrastin,6,7,3′,4′-tetramethoxyisoflavone, fujikenetin, dalpatein, milidurone,retusin, 8-methylretusin and their conjugates (The Flavonoids: Advancesin Research Chapman and Hall Ltd, 1982 edited by J. B. Harborne and T.J. Mabry—chapter Isoflavonoids P. M. Dewick pp 535-632).

[0034] “Total isoflavone levels” refers to all the isoflavones producedby an isoflavonoid-producing plant.

[0035] The term “isoflavonoid-producing plant” refers to a plant inwhich isoflavonoids naturally occur. Examples of isoflavonoid-producingplants include, but are not limited to, soybean, clover, mung bean,lentil, hairy vetch, alfalfa, lupine, sugar beet, and snow pea. In amore preferred embodiment, the preferred isoflavonoid-producing plantwould be soybean. Examples of other isoflavonoid-producing plants can befound in WO 93/23069, published Nov. 25, 1993, the disclosure of whichis hereby incorporated by reference.

[0036] The term “chalcone reductase” or “deoxychalcone synthase” refersto the polypeptide or enzyme that, together with a chalcone synthase andNADPH as a cofactor acts in the formation of isoliquiritigenin which isthen isomerized to form liquiritigenin, the precursor to daidzein,glycitein, and the pterocarpan phytoalexins. Chalcone reductase isabbreviated CHR.

[0037] The terms “CHR construct”, “chalcone reductase construct”, and“plasmid AC23” are used interchangeably herein.

[0038] The terms “polynucleotide”, “polynucleotide sequence”, “nucleicacid sequence”, “nucleic acid fragment”, and “isolated nucleic acidfragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides(usually found in their 5′-monophosphate form) are referred to by asingle letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and“N”for any nucleotide.

[0039] The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

[0040] The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

[0041] Moreover, the skilled artisan recognizes that substantiallysimilar nucleic acid sequences encompassed by this invention are alsodefined by their ability to hybridize, under moderately stringentconditions (for example, 0.5×SSC, 0.1% SDS, 60° C.) with the sequencesexemplified herein, or to any portion of the nucleotide sequencesdisclosed herein and which are functionally equivalent to any of thenucleic acid sequences disclosed herein. Stringency conditions can beadjusted to screen for moderately similar fragments, such as homologoussequences from distantly related organisms, to highly similar fragments,such as genes that duplicate functional enzymes from closely relatedorganisms. Post-hybridization washes determine stringency conditions.One set of preferred conditions involves a series of washes startingwith 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set ofstringent conditions involves the use of higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions involves the use oftwo final washes in 0.1×SSC, 0.1% SDS at 65° C.

[0042] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. An“allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When all the alleles present at a given locus ona chromosome are the same that plant is homozygous at that locus. If thealleles present at a given locus on a chromosome differ that plant isheterozygous at that locus.

[0043] “Coding sequence” refers to a DNA sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

[0044] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro, J. K., and Goldberg, R. B.(1989) Biochemistry of Plants 15:1-82.

[0045] The “translation leader sequence” refers to a polynucleotidesequence located between the promoter sequence of a gene and the codingsequence. The translation leader sequence is present in the fullyprocessed mRNA upstream of the translation start sequence. Thetranslation leader sequence may affect processing of the primarytranscript to mRNA, mRNA stability or translation efficiency. Examplesof translation leader sequences have been described (Turner, R. andFoster, G. D. (1995) Mol. Biotechnol. 3:225-236).

[0046] The “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht, I. L., et al. (1989)Plant Cell 1:671-680.

[0047] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. An RNA transcript is referred toas the mature RNA when it is an RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA (mRNA)” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to and synthesized from a mRNA template using the enzymereverse transcriptase. The CDNA can be single-stranded or converted intothe double-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065). The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

[0048] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is regulated by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of regulating theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

[0049] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory Press: Cold SpringHarbor, 1989. Transformation methods are well known to those skilled inthe art and are described below.

[0050] “PCR” or “Polymerase Chain Reaction” is a technique for thesynthesis of large quantities of specific DNA segments, consists of aseries of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk,Conn.). Typically, the double stranded DNA is heat denatured, the twoprimers complementary to the 3′ boundaries of the target segment areannealed at low temperature and then extended at an intermediatetemperature. One set of these three consecutive steps is referred to asa cycle.

[0051] The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

[0052] The terms “recombinant construct”, “expression construct”,“chimeric construct”, “construct”, and “recombinant DNA construct” areused interchangeably herein. A recombinant construct comprises anartificial combination of nucleic acid fragments, e.g., regulatory andcoding sequences that are not found together in nature. For example, achimeric construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al., (1985) EMBO J.4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others.

[0053] The term “expression”, as used herein, refers to the productionof a functional end-product e.g., a mRNA or a protein (precursor ormature).

[0054] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0055] “Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

[0056] “Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence (seeVaucheret et al. (1998) Plant J. 16:651-659; and Gura (2000) Nature404:804-808). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. Recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication WO 99/53050 published on Oct. 21, 1999 and more recently,Applicants' assignee's own WO 02/00904 published on Jan. 3, 2002). Thisincreases the frequency of co-suppression in the recovered transgenicplants. Hairpin structures may contain the target RNA forming either thestem or the loop of the hairpin. Another variation describes the use ofplant viral sequences to direct the suppression, or “silencing”, ofproximal mRNA encoding sequences (PCT Publication WO 98/36083 publishedon Aug. 20, 1998). Both of these co-suppressing phenomena have not beenelucidated mechanistically, although genetic evidence has begun tounravel this complex situation (Elmayan et al. (1998) Plant Cell10:1747-1757).

[0057] The polynucleotide sequences used for suppression do notnecessarily have to be 100% complementary to the polynucleotidesequences found in the gene to be suppressed. For example, suppressionof all the subunits of the soybean seed storage protein β-conglycininhas been accomplished using a polynucleotide derived from a portion ofthe gene encoding the α subunit (U.S. Pat. No. 6,362,399). β-conglycininis a heterogeneous glycoprotein composed of varying combinations ofthree highly negatively charged subunits identified as α, α′ and β. Thepolynucleotide sequences encoding the α and α′ subunits are 85%identical to each other while the polynucleotide sequences encoding theβ subunit are 75 to 80% identical to the α and α′ subunits. Thus,polynucleotides that are at least 75% identical to a region of thepolynucleotide that is target for suppression have been shown to beeffective in suppressing the desired target. The polynucleotide shouldbe at least 80% identical, preferably at least 90% identical, mostpreferably at least 95% identical, or the polynucleotide may be 100%identical to the desired target.

[0058] The present invention includes a method for decreasing the ratioof liquiritigenin-derived isoflavones relative to total isoflavonelevels in an isoflavonoid-producing plant the method comprising: a)transforming a plant cell with a recombinant construct comprising apromoter operably linked to a nucleic acid sequence of at least 200nucleotides having at least 75% sequence identity to SEQ ID NO:4; b)regenerating a transformed plant from the transformed plant cell of (a);and c) evaluating the transformed plant obtained from step (b) for areduced ratio of liquiritigenin-derived isoflavones relative to totalisoflavone levels as compared to the ratio of liquiritigenin-derivedisoflavones relative to total isoflavone levels in an untransformedplant.

[0059] Examples of isoflavonoid-producing plants which can be used topractice the invention include, but are not limited to, soybean, clover,mung bean, lentil, hairy vetch, alfalfa, lupine, sugar beet, and snowpea.

[0060] Any promoter can be used in accordance with the method of theinvention. Thus, the origin of the promoter chosen to drive expressionof the coding sequence is not important as long as it has sufficienttranscriptional activity to accomplish the invention by expressingtranslatable mRNA for the desired nucleic acid fragments in the desiredhost tissue. Either heterologous or non-heterologous (i.e., endogenous)promoters can be used to practice the invention. The promoter for use inthe present invention may be selected from the group consisting of aseed-specific promoter, root-specific promoter, vacuole-specificpromoter, and an embryo-specific promoter.

[0061] Among the most commonly used promoters are the nopaline synthase(NOS) promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. U.S.A.84:5745-5749), the octapine synthase (OCS) promoter, caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S promoter(Lawton et al. (1987) Plant Mol. Biol. 9:315-324), the CaMV 35S promoter(Odell et al. (1985) Nature 313:810-812), and the figwort mosaic virus35S promoter, the light inducible promoter from the small subunit ofrubisco, the Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 84:6624-66280, the sucrose synthase promoter (Yang et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87:4144-4148), the R gene complex promoter(Chandler et al. (1989) Plant Cell 1:1175-1183), the chlorophyll a/bbinding protein gene promoter, etc. Other commonly used promoters are,the promoters for the potato tuber ADPGPP genes, the sucrose synthasepromoter, the granule bound starch synthase promoter, the glutelin genepromoter, the maize waxy promoter, Brittle gene promoter, and Shrunken 2promoter, the acid chitinase gene promoter, and the zein gene promoters(15 kD, 16 kD, 19 kD, 22 kD, and 27 kD; Perdersen et al. (1982) Cell29:1015-1026). A plethora of promoters is described in WO 00/18963,published on Apr. 6, 2000, the disclosure of which is herebyincorporated by reference.

[0062] Examples of a seed-specific promoter include, but are not limitedto, the promoter for β-conglycinin (Chen et al. (1989) Dev. Genet 10:112-122), the napin promoter, and the phaseolin promoter. Othertissue-specific promoters that may be used to accomplish the inventioninclude, but are not limited to, the chloroplast glutamine synthase(GS2) promoter (Edwards et al. (1990) Proc. Natl. Acad. Sci. U.S.A.87:3459-3463), the chloroplast fructose-1,6-biophosphatase promoter(Lloyd et al. (1991) Mol. Gen. Genet. 225:209-2216), the nuclearphotosynthetic (ST-LS1) promoter (Stockhaus et al. (1989) EMBO J.8:2445-2451), the serine/threonine kinase (PAL) promoter, theglucoamylase promoter, the promoters for the Cab genes (cab6, cab-1, andcab-1R, Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778; Fejes etal. (1990) Plant Mol. Biol. 15:921-932; Lubberstedt et al. (1994) PlantPhysiol. 104:997-1006; Luan et al. (1992) Plant Cell 4:971-981), thepyruvate orthophosphate dikanase promoter (Matsuoka et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:9586-9590), the LhcB promoter (Cerdan et al.(1997) Plant Mol. Biol. 33:245-255), the PsbP promoter (Kretsch et al.(1995) Plant Mol. Biol. 28:219-229), the SUC2 sucrose H+ symporterpromoter (Truernit et al. (1995) Planta 196:564-570), and the promotersfor the thylakoid membrane genes (psaD, psaF, psaE, PC, FNR, atpC,atpD), etc..

[0063] The promoter is then operably linked, in a sense or antisenseorientation, using conventional means well known to those skilled in theart to a nucleic acid sequence of at least 200 nucleotides having atleast 75% sequence identity to SEQ ID NO:4.

[0064] Suppression in general is described above. However, in apreferred embodiment, the recombinant construct comprises a stem-loopstructure as described in PCT publication WO 02/00904, published Jan. 3,2002. WO 02/00904 discloses that suitable nucleic acid sequences andtheir reverse complement can be used to alter the expression of anyhomologous, endogenous RNA (i.e., the target RNA) which is flanked bythe suitable nucleic acid sequence and its reverse complement. Thesuitable nucleic acid sequence and its reverse complement can beunrelated to any endogenous RNA in the host, can be transcribed for byany nucleic acid sequence in the genome of the host provided thatnucleic acid sequence is not transcribed to any target mRNA or anysequence that is substantially similar to the target mRNA, or can betranslated into a synthetic or non-naturally occurring polypeptide. Whatis presented in WO 02/00904 is a very efficient and robust approach toachieving single, or multiple, gene co-suppression using single plasmidtransformation. Such constructs are composed of promoters linked tomRNA(s) coding regions, or fragments thereof, that are targeted forsuppression, and short complementary sequences that are unrelated to thetargets. The complementary sequences can be oriented both 5′, both 3′,or on either side of the target sequence. The complementary sequencesare preferred to be about 40-50 nucleotides in length, or morepreferably 50-100 nucleotides in length, or most preferably at least orgreater than 100-300 nucleotides.

[0065] The complementary sequences are unrelated to the target, but cancome from any other source. Preferred embodiments of these sequencesinclude, but are not limited to, plant sequences, bacterial sequences,animal sequences, viral or phage sequences, or completely artificial,i.e. non-naturally occurring, sequences not known to occur in anyorganism. These complementary sequences can be synthesized usingconventional means well known to those skilled in the art. Non-naturallycomplementary regions which can be used to practice the inventioninclude, but are not limited to, a polynucleotide that may be translatedinto the polypeptide Glu Leu Val IIe Ser Leu IIe Val Glu Ser(“ELVISLIVES”; shown in SEQ ID NO:8).

[0066] Methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens, and obtaining transgenic plants have beenpublished, among others, for cotton (U.S. Pat. No. 5,004,863, U.S. Pat.No. 5,159,135); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No.5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al.(1996) Plant Cell Rep. 15:653-657, McKently et al. (1995) Plant CellRep. 14:699-703); papaya (Ling, K. et al. (1991) Bio/technology9:752-758); and pea (Grant et al. (1995) Plant Cell Rep. 15:254-258).For a review of other commonly used methods of plant transformation seeNewell, C. A. (2000) Mol. Biotechnol. 16:53-65. One of these methods oftransformation uses Agrobacterium rhizogenes (Tepfler, M. andCasse-Delbart, F. (1987) Microbiol. Sci. 4:24-28). Transformation ofsoybeans using direct delivery of DNA has been published using PEGfusion (PCT publication WO 92/17598), electroporation (Chowrira, G. M.et al. (1995) Mol. Biotechnol. 3:17-23; Christou, P. et al. (1987) Proc.Natl. Acad. Sci. U.S.A. 84:3962-3966), microinjection, or particlebombardment (McCabe, D. E. et. al. (1988) Bio/Technology 6:923; Christouet al. (1988) Plant Physiol. 87:671-674).

[0067] There are a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, (1988) In.:Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., SanDiego, Calif.). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

[0068] In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant DNA fragments and recombinant expressionconstructs and the screening and isolating of clones, (see for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press; Maliga et al. (1995) Methods in Plant MolecularBiology, Cold Spring Harbor Press; Birren et al. (1998) Genome Analysis:Detecting Genes, 1, Cold Spring Harbor, N.Y.; Birren et al. (1998)Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, N.Y.; PlantMolecular Biology: A Laboratory Manual, eds. Clark, Springer, N.Y.(1997)).

[0069] In still another aspect, this invention includes anisoflavonoid-producing plant made by any of the instant methods whereinthe plant has a reduced ratio of liquiritigenin-derived isoflavonesrelative to total isoflavone levels as compared to the ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsin an untransformed plant.

[0070] In still a further aspect, there is included anisoflavonoid-producing plant comprising in its genome a recombinantconstruct comprising a promoter operably linked to a nucleic acidsequence of at least 200 nucleotides and having at least 75% sequenceidentity to SEQ ID NO:4 wherein the plant has a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsas compared to the ratio of liquiritigenin-derived isoflavones relativeto total isoflavone levels in an untransformed plant.

[0071] Also within the scope of this invention are seeds or plant partsobtained from such transformed plants. Plant parts includedifferentiated and undifferentiated tissues, including but not limitedto, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, andvarious forms of cells and culture such as single cells, protoplasts,embryos, and callus tissue. The plant tissue may be in plant or inorgan, tissue or cell culture.

[0072] In a still further aspect, this invention includes anisoflavonoid-containing product having a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsobtained from the seeds or plant parts of the invention.

[0073] Methods for obtaining such products are well known to thoseskilled in the art. For example, in the case of soybean, such productscan be obtained in a variety of ways. Conditions typically used toprepare soy protein isolates have been described by [Cho, et al, (1981)U.S. Pat. No. 4,278,597; Goodnight, et al. (1978) U.S. Pat. No.4,072,670]. Soy protein concentrates are produced by three basicprocesses: acid leaching (at about pH 4.5), extraction with alcohol(about 55-80%), and denaturing the protein with moist heat prior toextraction with water. Conditions typically used to prepare soy proteinconcentrates have been described by Pass [(1975) U.S. Pat. No.3,897,574] and Campbell et al. [(1985) in New Protein Foods, ed. byAltschul and Wilcke, Academic Press, Vol. 5, Chapter 10, Seed StorageProteins, pp 302-338].

[0074] “Isoflavonoid-containing protein products” can be defined asthose items produced from seed or other plant part of a suitable plantwhich are used in feeds, foods and/or beverages. One of the plants thatmay be used to prepare isoflavonoid-containing protein products issoybean. “Soy protein products” can include, but are not limited to,those items listed in Table 1. “Soy protein products”. TABLE 1 SoyProtein Products Derived from Soybean Seeds^(a) Whole Soybean ProductsRoasted Soybeans Baked Soybeans Soy Sprouts Soy Milk Specialty SoyFoods/Ingredients Soy Milk Tofu Tempeh Miso Soy Sauce HydrolyzedVegetable Protein Whipping Protein Processed Soy Protein Products FullFat and Defatted Flours Soy Grits Soy Hypocotyls Soybean Meal Soy MilkSoy Protein Isolates Soy Protein Concentrates Textured Soy ProteinsTextured Flours and Concentrates Textured Concentrates Textured Isolates

[0075] “Processing” refers to any physical and chemical methods used toobtain the products listed in Table 1 and includes, but is not limitedto, heat conditioning, flaking and grinding, extrusion, solventextraction, or aqueous soaking and extraction of whole or partial seeds.Furthermore, “processing” includes the methods used to concentrate andisolate soy protein from whole or partial seeds, as well as the varioustraditional Oriental methods in preparing fermented soy food products.Trading Standards and Specifications have been established for many ofthese products (see National Oilseed Processors Association Yearbook andTrading Rules 1991-1992). Products referred to as being “high protein”or “low protein” are those as described by these StandardSpecifications. “NSI” refers to the Nitrogen Solubility Index as definedby the American Oil Chemists' Society Method Ac4 41. “KOH NitrogenSolubility” is an indicator of soybean meal quality and refers to theamount of nitrogen soluble in 0.036 M KOH under the conditions asdescribed by Araba and Dale [(1990) Poult. Sci. 69:76-83]. “White”flakes refer to flaked, dehulled cotyledons that have been defatted andtreated with controlled moist heat to have an NSI of about 85 to 90.This term can also refer to a flour with a similar NSI that has beenground to pass through a No. 100 U.S. Standard Screen size. “Cooked”refers to a soy protein product, typically a flour, with an NSI of about20 to 60. “Toasted” refers to a soy protein product, typically a flour,with an NSI below 20. “Grits” refer to defatted, dehulled cotyledonshaving a U.S. Standard screen size of between No. 10 and 80. “SoyProtein Concentrates” refer to those products produced from dehulled,defatted soybeans by three basic processes: acid leaching (at about pH4.5), extraction with alcohol (about 55-80%), and denaturing the proteinwith moist heat prior to extraction with water. Conditions typicallyused to prepare soy protein concentrates have been described by Pass[(1975) U.S. Pat. No. 3,897,574; Campbell et al., (1985) in New ProteinFoods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10,Seed Storage Proteins, pp 302-338]. “Extrusion” refers to processeswhereby material (grits, flour or concentrate) is passed through ajacketed auger using high pressures and temperatures as a means ofaltering the texture of the material. “Texturing” and “structuring”refer to extrusion processes used to modify the physical characteristicsof the material. The characteristics of these processes, includingthermoplastic extrusion, have been described previously [Atkinson (1970)U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods, ed. byAltschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp 367-414].Moreover, conditions used during extrusion processing of complexfoodstuff mixtures that include soy protein products have been describedpreviously [Rokey (1983) Feed Manufacturing Technology III, 222-237;McCulloch, U.S. Pat. No. 4,454,804].

[0076] Also, within the scope of this invention are food, foodsupplements, food bars, and beverages that have incorporated therein anisoflavonoid-containing product of the invention. The beverage can be ina liquid or in a dry powdered form.

[0077] The isoflavonoid-containing product made according to the processof the present invention can be incorporated into a wide variety of foodand beverage applications. For example, it can be integrated into meatssuch as ground meats, emulsified meats, marinated meats, and meatsinjected with the soy product of the invention; it can also beincorporated into nutritional supplements; beverages such as nutritionalbeverages, sports beverages, protein fortified beverages, juices, milk,milk alternatives, and weight loss beverages; cheeses such as hard andsoft cheeses, cream cheese, and cottage cheese; frozen desserts such asice cream, ice milk, low fat frozen desserts, and non-dairy frozendesserts; yogurts; soups; puddings; bakery products; and saladdressings; and dips and spreads such as mayonnaise; and chip dips; andfood bars.

[0078] The isoflavonoid-containing product of the invention may also beincorporated into a cereal food product, a snack food product, a bakedgood product, a fried food product, a health food product, an infantformula, a beverage, a nutritional supplement, a dairy product, a petfood product, or animal feed.

[0079] A cereal food product is a food product derived from theprocessing of a cereal grain. A cereal grain includes any plant from thegrass family that yields an edible grain (seed). The most popular grainsare barley, corn, millet, oats, quinoa, rice, rye, sorghum, triticale,wheat and wild rice. Examples of a cereal food product include, but arenot limited to, whole grain, crushed grain, grits, flour, bran, germ,breakfast cereals, extruded foods, pastas, and the like.

[0080] A baked good product comprises any of the cereal food productsmentioned above and has been baked or processed in a manner comparableto baking, i.e., to dry or harden by subjecting to heat. Examples of abaked good product include, but are not limited to bread crumbs, bakedsnacks, mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels.

[0081] A snack food product comprises any of the above or belowdescribed food products.

[0082] A fried food product comprises any of the above or belowdescribed food products that has been fried.

[0083] A health food product is any food product that imparts a healthbenefit. Many oilseed-derived food products may be considered as healthfoods.

[0084] The beverage can be in a liquid or in a dry powdered form.

[0085] For example, there can be mentioned non-carbonated drinks;carbonated drinks; fruit juices, fresh, frozen, canned or concentrate;still or sparkling water; flavored or plain milk drinks, etc. Adult andinfant nutritional formulas are well known in the art and commerciallyavailable (e.g., Similac®, Ensure®, Jevity®, and Alimentum® from RossProducts Division, Abbott Laboratories).

[0086] Infant formulas are liquids or reconstituted powders fed toinfants and young children. They serve as substitutes for human milk.Infant formulas have a special role to play in the diets of infantsbecause they are often the only source of nutrients for infants.Although breast-feeding is still the best nourishment for infants,infant formula is a close enough second that babies not only survive butthrive. Infant formula is becoming more and more increasingly close tobreast milk.

[0087] A dairy product is a product derived from milk. These productsinclude, but are not limited to, whole milk, skim milk, fermented milkproducts such as yogurt or sour milk, cream, butter, condensed milk,dehydrated milk, coffee whitener, ice cream, cheese, whey products,lactose, etc.

[0088] In still another aspect this invention concerns a method ofproducing an isoflavonoid-containing product which comprises: (a)cracking the seeds obtained from transformed plants of the invention toremove the meats from the hulls; and (b) flaking the meats obtained instep (a) to obtain the desired flake thickness.

EXAMPLES

[0089] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

Example 1 Construction of Plasmids for Transformation of Glycine MAX

[0090] The ability to alter the ratio of individual isoflavonoids intransgenic plants was tested. For this purpose, a vector (plasmid AC23)was prepared that would be capable of suppressing chalcone reductase.

[0091] Preparation of Plasmid AC23

[0092] Plasmid AC23 contains a seed-specific expression promoterfollowed by nucleotides that promote formation of a stem loop structureflanking nucleotides encoding a portion of a soybean chalcone reductase,and followed by a transcription termination signal. It is wellunderstood by those skilled in the art that other sequences commonlyused in molecular manipulations may be used here. These sequences mayinclude any seed-specific promoter, any structure that promotesstem-loop formation, any polynucleotide encoding any portion of the geneor genes of interest inserted in sense or anti-sense orientation withrespect to the promoter and stem-loop structure, and any terminationsignal. It is also well known by those skilled in the art thatnucleotides promoting stem-loop formation are not always required forgene suppression. Plasmid AC23 was prepared as follows:

[0093] A polynucleotide encoding a portion of a soybean chalconereductase was inserted in the seed-specific expression vector pKS151 toobtain plasmid AC23. Vector pKS151 is depicted in FIG. 2 and itsnucleotide sequence is shown in SEQ ID NO:1. This vector has beendescribed in PCT Publication WO 02/00904, published Jan. 03, 2002, andis derived from the commercially available vector pSP72 (Promega,Madison, Wis.).

[0094] Vector pSP72 was modified as follows to produce plasmid pKS151:

[0095] a) deleting the polynucleotide fragment corresponding to the betalactamase coding region (nucleotides 1135 through 1995 corresponding);

[0096] b) inserting a polynucleotide fragment encoding HPT under thecontrol of the T7 promoter and termination signals, for expression ofthe HPT enzyme in bacteria;

[0097] c) adding a polynucleotide consisting essentially of the CaMV 35Spromoter/HPT/NOS 3′ for constitutive expression of the HPT enzyme inplants; and

[0098] d) adding a polynucleotide consisting essentially of a unique Not1 restriction endonuclease site surrounded by nucleotides that promoteformation of a stem structure which are flanked by the KTi promoter andKTi 3′ terminator.

[0099] Expression of HPT by two different promoters allows the selectionfor growth in the presence of hygromycin in bacterial and plant systems.The gene encoding the kunitz trypsin inhibitor 3 (KTi3) has beendescribed (Jofuku and Goldberg (1989) Plant Cell 1:1079-1093). Inplasmid pKS151 the KTi3 promoter includes about 2088 nucleotidesupstream (5′) from the translation initiation codon, and the KTi3terminator includes about 202 nucleotides downstream (3′) from thetranslation stop codon of KTi 3. Between the KTi3 5′ and 3′ regions is aunique Not I restriction endonuclease site. The Not I site is flanked bynucleotides that promote formation of a “stem-loop” structure when apolynucleotide from the gene of interest is inserted at the Not I site.The nucleotides promoting the formation of a stem are shown in SEQ IDNO:7. This “stem-loop” structure will have the polynucleotides from thegene of interest forming the loop. The stem structure is formed by twocopies of 36 nucleotides at the 5′ end of the Not I site and an invertedrepeat of the same two 36-nucleotide copies at the 3′ end.

[0100] Sequences encoding a portion of a soybean chalcone reductase wereinserted in the Not I site of pKS151 to create plasmid AC23. Thefragment corresponding to a portion of the chalcone reductase codingsequence was obtained by PCR amplification using clone src3c.pk009.e4 astemplate and primers chalcone reductase-Not1-sense (shown in SEQ IDNO:2) and chalcone reductase-Not1-antisense (shown in SEQ ID NO:3). Thenucleotide sequence of the cDNA insert in clone src3c.pk009.e4 is shownin SEQ ID NO:4. (SEQ ID NO:2) 5′-GCG GCC GCA TGG CTG CTG CTA TTG AAATC-3′ (SEQ ID NO:3) 5″-GCG GCC GCC CTG CTC GCA CCT TTC CTC AG-3′

[0101] The amplification reaction was performed using advantage 2polymerase and GC melt reagent (1 mM final concentration) and followingthe manufacturer's (Clontech, Palo Alto, Calif.) protocol. The resultingamplified DNA fragment was first cloned into TopoTA vector (Invitrogen,Carlsbad, Calif.). The fragment was liberated from the TopoTA vector byNot I digestion and was purified from an agarose gel using Qiagen GelPurification Kit (Qiagen, Valencia, Calif.). The purified DNA fragmentwas inserted into the Not I site of vector pKS151 to produce the plasmidAC23.

Example 2 Transformation of Somatic Soybean Embryo Cultures andRegeneration of Soybean Plants

[0102] The ability to increase the isoflavonoid levels in transgenicsoybean plants was tested by transforming soybean somatic embryocultures with plasmid AC23, selecting transformants that grew in thepresence of hygromycin, allowing plants to regenerate, and measuring thelevels of isoflavone produced in seeds.

[0103] Soybean embryogenic suspension cultures were transformed withplasmid AC23 by the method of particle gun bombardment.

[0104] The following stock solutions and media were used fortransformation and regeneration of soybean plants:

[0105] Stock Solutions (Per Liter):

[0106] MS Sulfate 100x stock: 37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86g ZnSO₄.7H₂O, 0.0025 g CuSO₄.5H₂O.

[0107] MS Halides 100x stock: 44.0 g CaCl₂.2H₂O, 0.083 g KI, 0.00125 gCOCl₂.6H₂O, 17.0 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g Na₂MoO₄.2H₂O, 3,724 gNa₂EDTA, 2.784 g FeSO₄.7H₂O.

[0108] B5 Vitamin stock: 100.0 g myo-inositol, 1.0 g nicotinic acid, 1.0g pyridoxine HCl, 10.0 g thiamine.

[0109] 2,4-D stock: 10 mg/mL

[0110] Media (per Liter):

[0111] SB55: 10 mL of each MS stock, 1 mL of B5 Vitamin stock, 0.8 gNH₄NO₃, 3.033 g KNO₃, 1 mL 2,4-D stock, 0.667 g asparagine, pH 5.7.

[0112] SB103: 1 pk. Murashige & Skoog salt mixture (Gibco, Carlsbad,Calif.), 60 g maltose, 2 g gelrite, pH 5.7.

[0113] SB71-1: B5 salts, 1 ml B5 vitamin stock, 30 g sucrose, 750 mgMgCl2, 2 g gelrite, pH 5.7.

[0114] Soybean (of the Jack variety) embryogenic suspension cultureswere maintained in 35 mL SB55 liquid media on a rotary shaker (150 rpm)at 28° C. with a mix of fluorescent and incandescent lights providing a16 hour day, 8 hour night cycle. Cultures were subcultured every 2 to 3weeks by inoculating approximately 35 mg of tissue into 35 mL of freshliquid media.

[0115] Soybean embryonic suspension cultures were transformed by themethod of particle gun bombardment (see Klein et al. (1987) Nature327:70-73) using a DuPont Biolistic PDS1000/He instrument. Embryos werebombarded with plasmid pAC23 in a 1:10 molar ratio. Transformed lineswere selected on medium containing hygromycin, and the presence ofplasmid pAC23 was determined by PCR. Transgenic plants were generatedfrom lines positive for the desired recombinant DNA fragments.

[0116] For bombardment, 5 μL of 1 μg/μL plasmid pAC23 DNA, 50 μL 2.5 MCaCl₂, and 20 μL 0.1 M spermidine were added to 50 μL of a 60 mg/mL 0.6μm gold particle suspension. The particle preparation was agitated for 3minutes, spun in a microfuge for 10 seconds and the supernatant removed.The DNA-coated gold particles were then washed once with 400 μL of 100%ethanol, resuspended in 40 μL of anhydrous ethanol, and sonicated threetimes for 1 second each. Five μL of the DNA-coated gold particles wasthen loaded on each macro carrier disk. Approximately 300 to 400 mg oftwo-week-old suspension culture was placed in an empty 60 mm×15 mm petridish and the residual liquid removed from the tissue using a pipette.The tissue was placed about 3.5 inches away from the retaining screenand bombarded twice. Membrane rupture pressure was set at 1100 psi andthe chamber was evacuated to −28 inches of Hg. Two plates were bombardedfor each experiment and, following bombardment, the tissue was dividedin half, placed back into liquid media, and cultured as described above.

[0117] Eleven days after bombardment, the liquid media was exchangedwith fresh SB55 media containing 50 mg/mL hygromycin. The selectivemedia was refreshed weekly. Seven weeks post bombardment, green,transformed tissue was observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue was removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Thus, each new line was treated as anindependent transformation event. Soybean suspension cultures can bemaintained as suspensions of embryos clustered in an immaturedevelopmental stage through subculture or can be regenerated into wholeplants by maturation and germination of individual somatic embryos.

[0118] Transformed embryogenic clusters were removed from liquid cultureand placed on SB103 solid agar media containing no hormones orantibiotics. Embryos were cultured for eight weeks at 26° C. with mixedfluorescent and incandescent lights on a 16 hour day, 8 hour nightschedule. During this period, individual embryos were removed from theclusters and analyzed at various stages of embryo development. Linesselected for hygromycin resistance were assayed by PCR for the presenceof the CHR construct contained in plasmid pAC23. The presence of the CHRconstruct was assayed by amplifying plant tissue using Primer 3 (shownin SEQ ID NO:5) and Primer 4 (shown in SEQ ID NO:6) (SEQ ID NO:5) primer3: 5′-CAC GGG ACG GAT GGT AGC AAC A-3′ (SEQ ID NO:6) primer 4: 5′-CCGATT CTC CCA ACA TTG CTT ATT C-3′

[0119] Somatic embryos became suitable for germination after eight weeksand were then removed from the maturation medium and dried in emptypetri dishes for 1 to 5 days. The dried embryos were then planted inSB71-1 medium where they were allowed to germinate under the samelighting and germination conditions described above. Germinated embryoswere transferred to sterile soil and grown to maturity. Seeds wereharvested.

Example 3 Analysis of Isoflavone Levels in Seeds of TransformantsContaining the CHR Construct

[0120] The quantity of isoflavones in seeds from transgenic plantscomprising the CHR construct was assayed. Seeds were ground, thecombined powder was extracted with methanol, hydrolyzed with base, andanalyzed. Base hydrolysis converts both malonylglucoside conjugates andacetylglucoside conjugates into glucoside conjugates (genistin, daidzin,and glycitin). Total glucoside conjugates are then measured. Whileaglycones are not measured by this method the amount of aglyconespresent is in such low quantities as to not affect the final results.The isoflavone numbers are reported as parts-per-million (ppm) ofglucoside conjugates in soybean. A more detailed explanation of thepreparation of seed extracts and measurement of isoflavones follows.

[0121] Five to eight seeds per transformant were combined and the seedswere ground to a fine powder using a single seed grinder set to thefinest setting. One gram of ground soybean seeds was extracted with 40mL MeOH:water (80:20 v/v) in a 125-mL Erlenmeyer flask at 65° C. on anorbital shaker. After shaking for 2 hours the flask was removed from theshaker and allowed to cool to room temperature. Three mL of 2N NaOH werethen added and the flask was returned to an orbital shaker at roomtemperature for 10 min. The flask was then removed from the shaker and1-mL glacial acetic acid was added. The sample was diluted to 50 mL withMeOH:water (80:20 v/v) and filtered through 5 pM filter paper in afunnel into another 125-mL Erlenmeyer flask. A mixture of 2.5 mL ofsample and 2.5 mL of MeOH:water (80:20 v/v) were diluted with water to10 mL in a volumetric flask. Particulate material was removed from asample of 1-1.5 mL by spinning in a microfuge tube, the liquidtransferred to a labeled autosampler vial, and analyzed by HPLC usingthe gradient indicated on Table 2 below. TABLE 2 HPLC Gradient SettingsTime Flow 1% acetic acid 1% acetic acid (min) (mL/min) in water (mL) inacetonitrile (mL) Initial 1.0 90 10  5.0 1.0 90 10 11.0 1.0 78 22 12.02.0 0 100 14.5 2.0 0 100 14.6 2.0 90 10 16.5 1.0 90 10

[0122] The HPLC was set to continue acquiring data to 17 minutes. Degasmode was set to continuous, column temperature was set to 30° C., andsample temperature was set to 4° C. The ultraviolet detector was set asfollows: sampling rate=10, wavelength=262 nm, autozero=0.1 minutes. Theamounts of isoflavones present were determined by comparing the resultsto a 5-point standard curve conducted using commercially availabledaidzin, glycitin, and genistin.

[0123] Analysis of R1 Seed from Transformation Events with Plasmid AC23

[0124] The levels of isoflavones in R1 seed from 88 plants derived from46 independent transformation events were assayed. These plants allcontained plasmid AC23. Each assay was performed using 5 to 8 seeds fromeach transformed plant as described above. Table 3 presents the level ofisoflavone components (daidzin, glycitin, or genistin), the sum of allisoflavones (total), as well as the sum of daidzin and glycitin (D+Gy),of samples of transgenic seeds positive for the CHR construct.Isoflavone levels are reported as parts-per-million (ppm). TABLE 3Isoflavone and Isoflavone Component Levels in R1 Seed Transgenic forPlasmid AC23 Daidzin Glycitin Genistin D + Gy Total Plant No. (ppm)(ppm) (ppm) (ppm) (ppm) 3058-2-1-1 587 370 845 957 1802 3058-2-1-2 817511 1241 1328 2569 3058-2-2-1 652 467 971 1119 2090 3058-2-2-2 444 358922 802 1725 3058-2-3-1 763 458 1277 1221 2498 3058-2-4-1 824 724 13721548 2921 3058-2-4-2 602 532 1278 1134 2412 3058-2-4-3 979 714 1969 16933661 3058-2-5-1 468 516 1186 984 2170 3058-2-5-2 552 623 1631 1175 28063058-3-1-1 1139 731 1763 1870 3632 3058-3-1-2 1000 753 1537 1753 32893058-3-2-1 936 1040 1508 1976 3483 3058-3-2-2 1099 1074 1667 2173 38413058-3-3-1 1640 869 2903 2509 5413 3058-3-3-2 1132 726 1753 1858 36113058-3-4-1 815 592 1193 1407 2600 3058-3-4-2 1092 631 1571 1723 32943058-3-6-2 1126 1063 2069 2189 4258 3058-3-6-3 785 888 2465 1673 41373058-3-8-1 298 250 1828 548 2376 3058-3-8-2 726 497 2405 1223 36273058-4-1-1 505 649 1342 1154 2495 3058-4-1-2 582 638 1494 1220 27133058-4-2-1 632 495 1408 1127 2534 3058-4-2-2 558 479 1317 1037 23533058-4-2-3 495 503 1305 998 2303 3058-5-2-1 859 790 1454 1649 31033058-5-2-2 1064 697 1686 1761 3447 3058-6-3-1 391 358 827 749 15773058-6-3-2 390 390 744 780 1523 3058-6-2-1 848 767 1870 1615 34853058-6-2-2 546 405 1796 951 2748 3063-1-1-1 885 1103 1458 1988 34453063-1-1-2 1003 940 1628 1943 3572 3063-1-2-1 1344 873 2045 2217 42623063-1-2-2 1156 487 1902 1643 3544 3063-1-4-1 523 643 884 1166 20503063-1-5-1 401 275 2619 676 3295 3063-1-5-2 458 518 1643 976 26193063-1-6-1 849 602 1660 1451 3111 3063-1-6-3 1333 750 2565 2083 46483063-1-7-2 168 235 1823 403 2226 3063-1-7-3 116 174 1270 290 15603063-2-10-1 202 338 1276 540 1815 3063-2-10-2 218 339 1590 557 21473063-2-1-1 468 390 936 858 1794 3063-2-11-2 791 513 1457 1304 27613063-2-12-1 299 293 1679 592 2271 3063-2-12-2 342 366 1647 708 23553063-2-12-3 364 343 2243 707 2950 3063-2-4-2 555 467 1166 1022 21883063-2-5-1 843 697 1920 1540 3459 3063-2-5-2 677 745 1653 1422 30743063-2-6-3 476 202 1258 678 1937 3063-2-8-1 1481 667 3005 2148 51533063-2-8-2 1070 671 2193 1741 3933 3063-2-9-1 425 495 1835 920 27553063-2-9-2 406 543 2080 949 3030 3063-3-1-1 474 699 1045 1173 22193063-3-1-2 654 565 1386 1219 2605 3063-3-2-1 525 595 923 1120 20433063-3-2-2 423 397 901 820 1722 3063-3-3-1 543 648 1029 1191 22203063-3-3-2 590 667 1204 1257 2461 3063-3-4-1 627 665 1732 1292 30243063-3-4-2 450 451 1268 901 2169 3063-3-5-1 530 347 1062 877 19383063-3-5-2 499 506 1232 1005 2237 3063-3-7-1 692 789 1623 1481 31043063-3-7-2 939 710 2102 1649 3751 3063-4-1-1 761 569 1256 1330 25853063-4-1-2 773 581 1402 1354 2756 3063-4-3-1 876 914 1390 1790 31813063-4-3-2 611 515 1102 1126 2228 3063-4-4-1 206 352 1668 558 22253063-4-4-3 187 493 622 680 1302 3063-4-5-1 588 744 884 1332 22163063-4-5-2 639 595 1174 1234 2408 3063-4-6-1 877 788 1686 1665 33523063-4-6-2 616 743 1180 1359 2540 3063-6-3-1 672 548 1301 1220 25213063-6-3-2 555 638 1022 1193 2215 3063-6-5-1 278 348 578 626 12043063-6-5-2 534 648 861 1182 2043 3063-6-7-1 498 357 2491 855 33463063-6-7-2 778 634 3065 1412 4477 3063-6-8-1 284 348 561 632 1193

[0125] The ration of the sum of daidzin and glycitin to totalisoflavones (D+Gy/T) was calculated for the same plants listed in Table3 and are listed in Table 4. FIG. 3 presents the results obtained forplants 1-44 and FIG. 4 presents the results obtained for plants 45-88.For ease of understanding, the plants from which the seeds are derivedare numbered 1 through 88 in the figures, and the plant number indicatedin the table. TABLE 4 Ratio of the Sum of Daidzin and Glycitin to TotalIsoflavones in R1 Seed Transgenic for Plasmid AC23 Plant Plant No. (D +Gy/T)100 1 3058-2-1-1 53.11 2 3058-2-1-2 51.69 3 3058-2-2-1 53.54 43058-2-2-2 46.49 5 3058-2-3-1 48.88 6 3058-2-4-1 53.00 7 3058-2-4-247.01 8 3058-2-4-3 46.24 9 3058-2-5-1 45.35 10 3058-2-5-2 41.87 113058-3-1-1 51.49 12 3058-3-1-2 53.30 13 3058-3-2-1 56.73 14 3058-3-2-256.57 15 3058-3-3-1 46.35 16 3058-3-3-2 51.45 17 3058-3-4-1 54.12 183058-3-4-2 52.31 19 3058-3-6-2 51.41 20 3058-3-6-3 40.44 21 3058-3-8-123.06 22 3058-3-8-2 33.72 23 3058-4-1-1 46.25 24 3058-4-1-2 44.97 253058-4-2-1 44.48 26 3058-4-2-2 44.07 27 3058-4-2-3 43.33 28 3058-5-2-153.14 29 3058-5-2-2 51.09 30 3058-6-3-1 47.50 31 3058-6-3-2 51.21 323058-6-2-1 46.34 33 3058-6-2-2 34.61 34 3063-1-1-1 57.71 35 3063-1-1-254.40 36 3063-1-2-1 52.02 37 3063-1-2-2 46.36 38 3063-1-4-1 56.88 393063-1-5-1 20.52 40 3063-1-5-2 37.27 41 3063-1-6-1 46.64 42 3063-1-6-344.81 43 3063-1-7-2 18.10 44 3063-1-7-3 18.59 45 3063-2-10-1 29.75 463063-2-10-2 25.94 47 3063-2-1-1 47.83 48 3063-2-11-2 47.23 493063-2-12-1 26.07 50 3063-2-12-2 30.06 51 3063-2-12-3 23.97 523063-2-4-2 46.71 53 3063-2-5-1 44.52 54 3063-2-5-2 46.26 55 3063-2-6-335.00 56 3063-2-8-1 41.68 57 3063-2-8-2 44.27 58 3063-2-9-1 33.39 593063-2-9-2 31.32 60 3063-3-1-1 52.86 61 3063-3-1-2 46.79 62 3063-3-2-154.82 63 3063-3-2-2 47.62 64 3063-3-3-1 53.65 65 3063-3-3-2 51.08 663063-3-4-1 42.72 67 3063-3-4-2 41.54 68 3063-3-5-1 45.25 69 3063-3-5-244.93 70 3063-3-7-1 47.71 71 3063-3-7-2 43.96 72 3063-4-1-1 51.45 733063-4-1-2 49.13 74 3063-4-3-1 56.27 75 3063-4-3-2 50.54 76 3063-4-4-125.08 77 3063-4-4-3 52.23 78 3063-4-5-1 60.11 79 3063-4-5-2 51.25 803063-4-6-1 49.67 81 3063-4-6-2 53.50 82 3063-6-3-1 48.39 83 3063-6-3-253.86 84 3063-6-5-1 51.99 85 3063-6-5-2 57.86 86 3063-6-7-1 25.55 873063-6-7-2 31.54 88 3063-6-8-1 52.98

[0126] As seen in the table above, the sum of daidzin and glycitin wasbetween 10% and 20% of the total isoflavones in two plants derived fromone transformation event positive for plasmid AC23. The sum of daidzinand glycitin was between 20% and 30% of the total isoflavones in eightplants derived from six independent transformation events positive forplasmid AC23. The sum of daidzin and glycitin was between 30% and 40% ofthe total isoflavones in eight plants from seven independenttransformation events positive for plasmid AC23.

[0127] The results shown in Table 4 and FIGS. 3 and 4 may be comparedwith those shown in Table 1 of Wang, C. et al. [(2000) J. Am. Oil Chem.Soc. 77:483-487]. The Wang et al. report shows analysis of the agronomiccharacteristics of 210 soybean cultivars grown in the State of SouthDakota in the United States and concludes that the isoflavone contentsin non-transgenic soybean plants varies greatly. Table 1 of the Wangreport shows the total isoflavones (in μg/g), and the total percent ofgenistein, daidzein, and glycitein for all 210 soybean cultivars.Addition of the total daidzein percent with the total glycitein percentfor each cultivar shows that it varies from a low of 35 (for GoldenHarvest H-1263 and Newton 1006) to a high of 54 (for Praire Brand227EXP). As shown in Table 4 and FIGS. 3 and 4 of the presentapplication, suppression of chalcone reductase results in transgenicplants having even lower levels of daidzin and glycitin than is reportedby Wang et al.. The isoflavone analyses in the present application wereperformed using samples containing from 5 to 8 R1 seeds. It is expectedthat a bulk sample of R1 seeds will contain a combination of wild-typeand transgenic seed with, on average, 1/4 of the seeds being wild-type.Thus, the levels of daidzin plus glycitin in the transgenic seeds alonewill be even lower than what is shown in Table 4 and FIGS. 3 and 4.

[0128] The data above shows that suppression of CHR inisoflavonoid-producing plants results in such plants having reducedlevels of liquiritigenin-derived isoflavones.

1 8 1 7701 DNA Artificial Sequence Expression Vector pKS151 1 cgcgcccgatcatccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt 60 agaggccccaaggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc 120 ctttcgggctttgttagcag ccggatcgat ccaagctgta cctcactatt cctttgccct 180 cggacgagtgctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt 240 ccagacggccgcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg 300 gacgattgcgtcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa 360 gctctgatagagttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc 420 tgcaagctccggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca 480 cggcctccagaagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct 540 ccagtcaatgaccgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc 600 cgcgtgcacgaggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag 660 agcctgcgcgacggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg 720 gggatcagcaatcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg 780 tccgaatgggccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc 840 ctccgcgaccggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac 900 accctgtgcacggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag 960 cacttccggaatcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta 1020 gaaaccatcggcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct 1080 gaaagcacgagattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt 1140 ttcgatcagaaacttctcga cagacgtcgc ggtgagttca ggcttttcca tgggtatatc 1200 tccttcttaaagttaaacaa aattatttct agagggaaac cgttgtggtc tccctatagt 1260 gagtcgtattaatttcgcgg gatcgagatc gatccaattc caatcccaca aaaatctgag 1320 cttaacagcacagttgctcc tctcagagca gaatcgggta ttcaacaccc tcatatcaac 1380 tactacgttgtgtataacgg tccacatgcc ggtatatacg atgactgggg ttgtacaaag 1440 gcggcaacaaacggcgttcc cggagttgca cacaagaaat ttgccactat tacagaggca 1500 agagcagcagctgacgcgta cacaacaagt cagcaaacag acaggttgaa cttcatcccc 1560 aaaggagaagctcaactcaa gcccaagagc tttgctaagg ccctaacaag cccaccaaag 1620 caaaaagcccactggctcac gctaggaacc aaaaggccca gcagtgatcc agccccaaaa 1680 gagatctcctttgccccgga gattacaatg gacgatttcc tctatcttta cgatctagga 1740 aggaagttcgaaggtgaagg tgacgacact atgttcacca ctgataatga gaaggttagc 1800 ctcttcaatttcagaaagaa tgctgaccca cagatggtta gagaggccta cgcagcaggt 1860 ctcatcaagacgatctaccc gagtaacaat ctccaggaga tcaaatacct tcccaagaag 1920 gttaaagatgcagtcaaaag attcaggact aattgcatca agaacacaga gaaagacata 1980 tttctcaagatcagaagtac tattccagta tggacgattc aaggcttgct tcataaacca 2040 aggcaagtaatagagattgg agtctctaaa aaggtagttc ctactgaatc taaggccatg 2100 catggagtctaagattcaaa tcgaggatct aacagaactc gccgtgaaga ctggcgaaca 2160 gttcatacagagtcttttac gactcaatga caagaagaaa atcttcgtca acatggtgga 2220 gcacgacactctggtctact ccaaaaatgt caaagataca gtctcagaag accaaagggc 2280 tattgagacttttcaacaaa ggataatttc gggaaacctc ctcggattcc attgcccagc 2340 tatctgtcacttcatcgaaa ggacagtaga aaaggaaggt ggctcctaca aatgccatca 2400 ttgcgataaaggaaaggcta tcattcaaga tgcctctgcc gacagtggtc ccaaagatgg 2460 acccccacccacgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 2520 agtggattgatgtgacatct ccactgacgt aagggatgac gcacaatccc actatccttc 2580 gcaagacccttcctctatat aaggaagttc atttcatttg gagaggacac gctcgagctc 2640 atttctctattacttcagcc ataacaaaag aactcttttc tcttcttatt aaaccatgaa 2700 aaagcctgaactcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt 2760 ctccgacctgatgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg 2820 agggcgtggatatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta 2880 tgtttatcggcactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga 2940 attcagcgagagcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga 3000 cctgcctgaaaccgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat 3060 cgctgcggccgatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg 3120 tcaatacactacatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg 3180 gcaaactgtgatggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat 3240 gctttgggccgaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa 3300 caatgtcctgacggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt 3360 cggggattcccaatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat 3420 ggagcagcagacgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct 3480 ccgggcgtatatgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa 3540 tttcgatgatgcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg 3600 gactgtcgggcgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt 3660 agaagtactcgccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata 3720 gtgaggtacctaaagaagga gtgcgtcgaa gcagatcgtt caaacatttg gcaataaagt 3780 ttcttaagattgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat 3840 tacgttaagcatgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt 3900 atgattagagtcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca 3960 aactaggataaattatcgcg cgcggtgtca tctatgttac tagatcgatg tcgaatctga 4020 tcaacctgcattaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 4080 cttccgcttcctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 4140 cagctcactcaaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 4200 acatgtgagcaaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 4260 ttttccataggctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 4320 ggcgaaacccgacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 4380 gctctcctgttccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 4440 gcgtggcgctttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 4500 ccaagctgggctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 4560 actatcgtcttgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 4620 gtaacaggattagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 4680 ctaactacggctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 4740 ccttcggaaaaagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 4800 gtttttttgtttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 4860 tgatcttttctacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 4920 tcatgacattaacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg 4980 gtgatgacggtgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 5040 aagcggatgccgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc 5100 ggggctggcttaactatgcg gcatcagagc agattgtact gagagtgcac catatggaca 5160 tattgtcgttagaacgcggc tacaattaat acataacctt atgtatcata cacatacgat 5220 ttaggtgacactatagaacg gcgcgccgtc gacggatata atgagccgta aacaaagatg 5280 attaagtagtaattaatacg tactagtaaa agtggcaaaa gataacgaga aagaaccaat 5340 ttctttgcattcggccttag cggaaggcat atataagctt tgattatttt atttagtgta 5400 atgatttcgtacaaccaaag catttattta gtactctcac acttgtgtcg cggccggagc 5460 tggtcatctcgctcatcgtc gagtcggcgg ccggagctgg tcatctcgct catcgtcgag 5520 tcggcggccgccgactcgac gatgagcgag atgaccagct ccggccgccg actcgacgat 5580 gagcgagatgaccagctccg gccgcttggg gggctatgga agactttctt agttagttgt 5640 gtgaataagcaatgttggga gaatcgggac tacttatagg ataggaataa aacagaaaag 5700 tattaagtgctaatgaaata tttagactga taattaaaat cttcacgtat gtccacttga 5760 tataaaaacgtcaggaataa aggaagtaca gtagaattta aaggtactct ttttatatat 5820 acccgtgttctctttttggc tagctagttg cataaaaaat aatctatatt tttatcatta 5880 ttttaaatatcttatgagat ggtaaatatt tatcataatt ttttttacta ttatttatta 5940 tttgtgtgtgtaatacatat agaagttaat tacaaatttt atttactttt tcattatttt 6000 gatatgattcaccattaatt tagtgttatt atttataata gttcatttta atctttttgt 6060 atatattatgcgtgcagtac ttttttccta catataacta ctattacatt ttatttatat 6120 aatatttttattaatgaatt ttcgtgataa tatgtaatat tgttcattat tatttcagat 6180 tttttaaaaatatttgtgtt attatttatg aaatatgtaa tttttttagt atttgatttt 6240 atgatgataaagtgttctaa attcaaaaga agggggaaag cgtaaacatt aaaaaacgtc 6300 atcaaacaaaaacaaaatct tgttaataaa gataaaactg tttgttttga tcactgttat 6360 ttcgtaatataaaaacatta tttatattta tattgttgac aaccaaattt gcctatcaaa 6420 tctaaccaatataatgcatg cgtggcaggt aatgtactac catgaactta agtcatgaca 6480 taataaaccgtgaatctgac caatgcatgt acctanctaa attgtatttg tgacacgaag 6540 caaatgattcaattcacaat ggagatggga aacaaataat gaagaaccca gaactaagaa 6600 agcttttctgaaaaataaaa taaaggcaat gtcaaaagta tactgcatca tcagtccaga 6660 aagcacatgatattttttta tcagtatcaa tgcagctagt tttattttac aatatcgata 6720 tagctagtttaaatatattg cagctagatt tataaatatt tgtgttatta tttatcattt 6780 gtgtaatcctgtttttagta ttttagttta tatatgatga taatgtattc caaatttaaa 6840 agaagggaaataaatttaaa caagaaaaaa agtcatcaaa caaaaaacaa atgaaagggt 6900 ggaaagatgttaccatgtaa tgtgaatgtt acagtatttc ttttattata gagttaacaa 6960 attaactaatatgattttgt taataatgat aaaatatttt ttttattatt atttcataat 7020 ataaaaatagtttacttaat ataaaaaaaa ttctatcgtt cacaacaaag ttggccacct 7080 aatttaaccatgcatgtacc catggaccat attaggtaac catcaaacct gatgaagaga 7140 taaagagatgaagacttaag tcataacaca aaaccataaa aaacaaaaat acaatcaacc 7200 gtcaatctgaccaatgcatg aaaaagctgc aatagtgagt ggcgacacaa agcacatgat 7260 tttcttacaacggagataaa accaaaaaaa tatttcatga acaacctaga acaaataaag 7320 cttttatataataaatatat aaataaataa aggctatgga ataatatact tcaatatatt 7380 tggattaaataaattgttgg cggggttgat atatttatac acacctaaag tcacttcaat 7440 ctcattttcacttaactttt attttttttt tctttttatt tatcataaag agaatattga 7500 taatatactttttaacatat ttttatgaca ttttttattg gtgaaaactt attaaaaatc 7560 ataaattttgtaagttagat ttatttaaag agttcctctt cttattttaa attttttaat 7620 aaatttttaaataactaaaa tttgtgttaa aaatgttaaa aaatgtgtta ttaacccttc 7680 tcttcgaggatccaagcttg g 7701 2 29 DNA Artificial Sequence Amplification primerchalcone reductase Not1 sense 2 gcggccgcat ggctgctgct attgaaatc 29 3 29DNA Artificial Sequence Amplification primer chalcone reductase Not1antisense 3 gcggccgccc tgctcgcacc tttcctcag 29 4 1410 DNA Glycine max 4acaagaagga tggtccaaaa gtatctaata ggccatctcg atctcatctg accaagcttc 60ctggttagtt cctttgaatt gaataatata aaaaaaagaa gatgatggat gtgggtagag 120ctcagtataa cccacctacc tccaattgct gacttttcaa aggccaaaca tgaagaaatg 180ttgcagtata aaaaggggtg cccttcagtt atgtccatca acaaatattg gaatactaca 240ctatacttgt caaccctttg agagttagaa tggctgctgc tattgaaatc cccacaatag 300tgtttccaaa ctcctctgcc caacagagga tgccagtggt tggaatggga tctgcccctg 360acttcacatg caagaaagac acaaaggagg ctatcattga ggccgtgaaa cagggttaca 420gacacttcga cactgctgct gcttatggct ctgaacaggc tctcggtgaa gctctcaagg 480aagctatcca tcttggcctc gtctcccgcc aagacctctt tgtcacttcc aagctttggg 540tcaccgaaaa tcatcctcat cttgtccttc ctgctttgcg caaatcactt aaaactcttc 600aactagagta cttggacctg tatctcatcc actggcccct gagttctcag ccagggaagt 660tctcatttcc aattgaagta gaagatctct tgccttttga cgtgaagggt gtgtgggaat 720ccatggaaga gtgccagaaa cttggcctca ccaaagccat tggagtcagc aacttctctg 780tcaagaagct tcagaatctg ctctctgttg ctaccatccg tcccgtggtc gatcaagtgg 840agatgaacct tgcatggcaa cagaagaagc taagagagtt ctgcaaagaa aatgggataa 900tagtgactgc gttctctcca ctgaggaaag gtgcgagcag gggcccaaat gaagtgatgg 960agaatgatgt gctgaaggag attgcagagg ctcatgggaa atccatagcc caggtgagtc 1020tgagatggtt gtacgaacaa ggtgtgacat ttgtgccaaa gagctacgat aaggagagga 1080tgaaccagaa tctgcacatc tttgactggg cattgactga acaagatcat cacaaaataa 1140gtcaaatcag ccagagccgt ttgatcagcg gacccaccaa accacaactc gctgatctct 1200gggatgatca aatataaact atttactact atgcagctcc cactctattt ttataatcca 1260tctttttacc tcttgtttca ttttacgttt aaataattca tgccatgcca cttcttattt 1320tagatttcac aatcaataaa ctaggcacgc gcggcacatg atatgaataa actatgttca 1380attttttttt caaaaaaaaa aaaaaaaaaa 1410 5 22 DNA Artificial SequenceAmplification primer Primer3 5 cacgggacgg atggtagcaa ca 22 6 25 DNAArtificial Sequence Amplification primer Primer4 6 ccgattctcc caacattgcttattc 25 7 154 DNA Artificial Sequence Artificial sequence containing aNotI site flanked by two 36-nucleotide repeats and having an EagI siteat each end. 7 cggccggagc tggtcatctc gctcatcgtc gagtcggcgg ccggagctggtcatctcgct 60 catcgtcgag tcggcggccg ccgactcgac gatgagcgag atgaccagctccggccgccg 120 actcgacgat gagcgagatg accagctccg gccg 154 8 10 PRTArtificial Sequence Translation of one 30 nucleotide repeat found inpKS151 8 Glu Leu Val Ile Ser Leu Ile Val Glu Ser 1 5 10

What is claimed is:
 1. A method for decreasing the ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsin an isoflavonoid-producing plant the method comprising: a)transforming a plant cell with a recombinant construct comprising apromoter operably linked to a nucleic acid sequence of at least 200nucleotides having at least 75% sequence identity to SEQ ID NO:4; b)regenerating a transformed plant from the transformed plant cell of (a);and c) evaluating the transformed plant obtained from step (b) for areduced ratio of liquiritigenin-derived isoflavones relative to totalisoflavone levels as compared to the ratio of liquiritigenin-derivedisoflavones relative to total isoflavone levels in an untransformedplant.
 2. The method of claim 1 wherein the promoter is operably linked,in a sense orientation, to the nucleic acid sequence.
 3. The method ofclaim 1 wherein the promoter is operably linked, in an anti-senseorientation, to the nucleic acid sequence.
 4. The method of claim 1wherein the recombinant construct comprises a stem-loop structure. 5.The method of claim 4 wherein the nucleic acid sequence forms a stem inthe stem-loop structure.
 6. The method of claim 4 wherein the nucleicacid sequence forms a loop in the stem-loop structure.
 7. The method ofclaim 4 wherein the nucleic acid sequence forms a loop in the stem-loopstructure and the stem consists essentially of SEQ ID NO:7.
 8. Themethod of claim 1 wherein the promoter is a seed-specific promoter. 9.The method of claim 1 wherein the isoflavonoid-producing plant isselected from the group consisting of soybean, clover, mung bean,lentil, hairy vetch, alfalfa, lupine, sugar beet, and snow pea.
 10. Anisoflavonoid-producing plant made by the method of any of claims 1 to 8wherein the plant has a reduced ratio of liquiritigenin-derivedisoflavones relative to total isoflavone levels as compared to the ratioof liquiritigenin-derived isoflavones relative to total isoflavonelevels in an untransformed plant.
 11. The isoflavonoid-producing plantof claim 9 wherein the plant is selected from the group consisting ofsoybean, clover, mung bean, lentil, hairy vetch, alfalfa, lupine, sugarbeet, and snow pea.
 12. Seeds or plant parts of the plant of claim 11.13. An isoflavonoid-containing product having a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsobtained from the seeds or plant parts of claim
 12. 14. Theisoflavonoid-containing product of claim 13 wherein the product isselected from the group consisting of protein isolate, proteinconcentrate, meal, grits, full fat and defatted flours, texturedproteins, textured flours, textured concentrates, and textured isolates.15. A food which has incorporated therein the isoflavonoid-containingproduct of claim
 13. 16. A nutritional supplement which has incorporatedtherein the isoflavonoid-containing product of claim
 13. 17. A food barwhich has incorporated therein the isoflavonoid-containing product ofclaim
 13. 18. A beverage which has incorporated therein theisoflavonoid-containing product of claim
 13. 19. Anisoflavonoid-containing protein product having a reduced ratio ofliquiritigenin-derived isoflavones relative to a total isoflavone levelobtained from the seeds of claim 12 wherein the seeds are soybean seeds.20. The isoflavonoid-containing protein product of claim 19 wherein theisoflavonoid product is selected from the group consisting of proteinisolate, protein concentrate, meal, grits, full fat and defatted flours,textured proteins, textured flours, textured concentrates, texturedisolates, soymilk, tofu, fermented soy products, and whole bean soyproducts.
 21. An isoflavonoid-producing plant comprising in its genome arecombinant construct comprising a promoter operably linked to a nucleicacid sequence of at least 200 nucleotides and having at least 75%sequence identity to SEQ ID NO:4 wherein the plant has a reduced ratioof liquiritigenin-derived isoflavones relative to total isoflavonelevels as compared to the ratio of liquiritigenin-derived isoflavonesrelative to total isoflavone levels in an untransformed plant.
 22. Theisoflavonoid-producing plant of claim 21 wherein the plant is selectedfrom the group consisting of soybean, clover, mung bean, lentil, hairyvetch, alfalfa, lupine, sugar beet, and snow pea.
 23. The plant of claim22 wherein recombinant construct comprises a promoter operably linked,in sense orientation, to the nucleic acid sequence.
 24. The plant ofclaim 22 wherein recombinant construct comprises a promoter operablylinked, in an anti-sense orientation, to the nucleic acid sequence. 25.The plant of claim 22 wherein the recombinant construct comprises astem-loop structure.
 26. The plant of claim 22 wherein the recombinantconstruct comprises a stem-loop structure in which the nucleic acidsequence forms the stem.
 27. The plant of claim 22 the recombinantconstruct comprises a stem-loop structure in which the nucleic acidsequence forms the loop.
 28. The plant of claim 22 wherein therecombinant construct comprises a stem-loop structure in which thenucleic acid sequence forms the loop in the stem-loop structure and thestem consists essentially of SEQ ID NO:7.
 29. The plant of claim 22wherein the recombinant construct comprises a seed-specific promoter.30. Seeds or plant parts of the plant of any of claims 22-29.
 31. Anisoflavonoid-containing product having a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsobtained from the seeds or plant parts of claim
 30. 32. Theisoflavonoid-containing product of claim 31 wherein the product isselected from the group consisting of protein isolate, proteinconcentrate, meal, grits, full fat and defatted flours, texturedproteins, textured flours, textured concentrates, and textured isolates.33. A food which has incorporated therein the isoflavonoid-containingproduct of claim
 31. 34. A nutritional supplement which has incorporatedtherein the isoflavonoid-containing product of claim
 31. 35. A food barwhich has incorporated therein the isoflavonoid-containing product ofclaim
 31. 36. A beverage which has incorporated therein theisoflavonoid-containing product of claim
 31. 37. Anisoflavone-containing protein product having a reduced ratio ofliquiritigenin-derived isoflavones relative to total isoflavone levelsobtained from the seeds of claim 31 wherein the seeds are soybean seeds.38. The isoflavonoid-containing protein product of claim 31 wherein theisoflavonoid product is selected from the group consisting of proteinisolate, protein concentrate, meal, grits, full fat and defatted flours,textured proteins, textured flours, textured concentrates, texturedisolates, soymilk, tofu, fermented soy products, and whole bean soyproducts.
 39. A method of producing an isoflavonoid-containing producthaving a reduced ratio of liquiritigenin-derived isoflavones relative tototal isoflavone levels which comprises: (a) cracking the seeds of claim12 or claim 30 to remove the meats from the hulls; and (b) flaking themeats obtained in step (a) to obtain the desired flake thickness.